Use of Dried Blood Smears for Detection of Feline Hemoplasmas Using Real-Time Polymerase Chain Reaction

Abstract

Abstract. The objective of the current study was to determine the sensitivity and specificity of real-time polymerase chain reaction (real-time PCR) for feline hemoplasmas when applied to DNA extracted from dried whole-blood smears in comparison to that for DNA extracted from liquid whole blood. Blood samples were collected into ethylenediamine tetra-acetic acid tubes from 305 cats with possible or suspected hemoplasmosis, and dried blood smears from each sample were prepared. DNA was extracted from blood smears and a 160-ml aliquot of each liquid blood sample by using a robotic extractor and was subjected to real-time PCR for feline glyceraldehyde-3-phosphate dehydrogenase (liquid blood), 18S ribosomal RNA (rRNA; dried blood), and ''Candidatus Mycoplasma haemominutum'', Mycoplasma haemofelis, and ''Candidatus Mycoplasma turicensis'' DNA. When using the results for liquid whole blood as the gold standard, the sensitivity of each assay for ''Ca. M. haemominutum'', M. haemofelis, and ''Ca. M. turicensis'' was 49 of 66 (74%), 11 of 13 (85%), and 11 of 20 (55%), respectively. The specificity of each assay was 224 of 234 (96%), 287 of 287 (100%), and 280 of 280 (100%), respectively. When possible, liquid blood samples should be submitted for detection of feline hemoplasmas by using real-time PCR. The improved sensitivity of real-time PCR on blood smears for M. haemofelis compared with that of the other hemoplasma species may reflect the higher organism burdens associated with infection with this species.

Investigation of human haemotropic Mycoplasma infections using a novel generic haemoplasma qPCR assay on blood samples and blood smears

“…A number of methods were tried (20 smears per method) to remove the blood from the slides for DNA extraction: use of a scalpel (Aubouy & Carme, 2004) or slide scraper (Sykes et al, 2008), as previously described, and use of a sterile swab moistened in PBS firmly rubbed over the entirety of the slide. Use of the scalpel or slide scraper made it difficult to remove all of the blood from the slide and concern regarding laboratory contamination arose due to blood smear material becoming airborne during removal, so an optimized method using a swab moistened in PBS was used for further studies.…”

Feline hemotropic mycoplasmas

“…Because the pathogenic potential of each hemoplasma species differs, the laboratory must be consulted to determine the species specificity of the assay(s) offered. Dried blood smears can also be used for PCR but are less sensitive than a larger volume of liquid whole blood 82 . Treatment with antimicrobial drugs may result in false negative results using PCR, so blood must be collected before initiating antimicrobial therapy.…”

Detection and identification of mycobacteria in fixed stained smears and formalin‐fixed paraffin‐embedded tissues using PCR

“…The present results are consistent with recent studies of haemotropic mycoplasmas (Sykes et al . , Tasker et al . , Barker et al .…”

“…Reports of using the polymerase chain reaction (PCR) to amplify haemoparasite DNA from blood films (Sykes et al . , Tasker et al . , Barker et al .…”